Logan Smesrud's blog

This week I tried to get some more answers from the mutants that I didn’t send to be sequenced last week. To prepare for Tuesday’s transformations, on Monday I made media and inoculated all of my remaining mutants and two controls for overnight cultures. Since Monday was just waiting for bacteria to grow, I had time to work on my final paper and presentation. My mentor offered to listen to my presentation and gave me some feedback. This was really helpful and also very kind since she has to hear it a few times anyways!

On Monday morning I took photos of my Carbon Source “Feeding” Experiment. I noticed that the biofilm in lignin appeared to be somewhat weaker than it was before and the biofilm in humic acids was much thicker. This observation lead me to believe that P.putida cannot consume any more lignin, but is still digesting the humic acids. My mentor and I noticed that the solution in the oxidizing culture of humic acids (with the thick biofilm) was a much clearer solution than the non-oxidizing mutant in humic acids.

On Monday I did not get as much information from my motility plates as I would’ve liked, but I did get a few good photos. There was denser and darker oxidation occurring next to certain carbon sources. More oxidation means that there are more cells, and if there are more cells, there must have been more growth in that area. Another way to observe the bacteria seeking and eating the carbon source was when there was a bulge in the circle towards a certain carbon source. This means that the bacteria made flagella so they could swim more towards the food source they could eat.

The culture tubes I started last week with various P.putida mutants growing in the presence of various carbon sources did not much change from when I checked them on Friday. This makes me wonder if maybe there is a limit on how much of the carbon source the bacteria are capable of breaking down and digesting.

The Carbon Source Experiment I started last week (many identical cultures growing and plating dilutions every couple of days) involves a lot of repetition repetition repetition. I start the day with counting colonies on the dilution plates from the other day and calculate colony forming units (CFU) per mL. Then I make all of the same dilutions as I did before, but with a different set of cultures (that have had more time to grow), and I plate two dilutions for each.

I can’t believe I’m already half way through my internship… It has gone by incredibly fast! On Monday I checked on my second round of motility plates and discovered that my mutants varied in their motility phenotype. This tells me that in all of my mutants, although the transposon suppressed the original mutation of the bacteria by restoring Mn oxidation, the transposon was inserted in different parts of the genome. I categorized my 28 mutants by Mn oxidation and motility phenotypes.

It turns out that streaking for single colonies of bacteria is even trickier than I thought! Monday is the third time I’ve streaked the mutants obtained from my conjugation experiments I did over the past few weeks. I now have 30 potential mutants where Mn oxidation has been restored, out of around a couple hundred thousand colonies of bacteria. In these oxidizing colonies, my mentor and I hypothesis that the transposon disrupted the gene encoding the negative regulator because of where it randomly inserted itself into the bacteria’s genome.

This week ended up being pretty short due to a family trip and the 4th of July holiday. I was out of town with my family on Monday and Tuesday. We stayed in Chiloquin, in the Klamath Basin, and fished all day in my dad’s drift boat on the Williamson River. I caught some trout, and it was a lot of fun! I was thankful that my mentor was understanding and flexible so that I could join my family on this trip. Since I was out of town Monday and Tuesday, Wednesday was a pretty busy day! Our to-do list for the day took up an entire sheet of paper, but we got it all done!

Wow, I can't believe another week has already gone by! On Monday I checked up on the bacteria I was growing on plates and in liquid, with various mutations, to observe the role of certain genes in manganese oxidation. The plates provided me with a much clearer conclusion than the liquid, because hardly any of the tubes showed Mn oxidation in liquid. We also continued with the conjugation experiment. Unfortunately it did not go exactly how we had planned.

Hello there! My name is Logan Smesrud, and I am an environmental engineering student at Oregon State University. I am a summer intern at CMOP through the Johnson Fellowship program, organized through my university. The Johnsons are alums of my engineering department and started this program specifically for chemical, biological, and environmental engineering students who have just finished their first year. Their mission is to give young undergraduates a taste of research and real work experience at the beginning of our undergraduate career.